Role of nitric oxide- and vasoactive intestinal polypeptide-containing neurones in human gastric fundus strip relaxations

Abstract

The morphological pattern and motor correlates of nitric oxide (NO) and vasoactive intestinal polypeptide (VIP) innervation in the human isolated gastric fundus was explored. By using the nicotinamide adenine dinucleotide phosphate hydrogen (NADPH)-diaphorase and specific rabbit polyclonal NO-synthase (NOS) and VIP antisera, NOS- and VIP-containing varicose nerve fibres were identified throughout the muscle layer or wrapping ganglion cell bodies of the myenteric plexus. NOS-immunoreactive (IR) neural cell bodies were more abundant than those positive for VIP-IR. The majority of myenteric neurones containing VIP coexpressed NADPH-diaphorase. Electrical stimulation of fundus strips caused frequency-dependent NANC relaxations. N(G)-nitro-L-arginine (L-NOARG: 300 microM) enhanced the basal tone, abolished relaxations to 0.3 - 3 Hz (5 s) and those to 1 Hz (5 min), markedly reduced ( approximately 50%) those elicited by 10 - 50 Hz, and unmasked or potentiated excitatory cholinergic responses at frequencies > or =1 Hz. L-NOARG-resistant relaxations were virtually abolished by VIP (100 nM) desensitization at all frequencies. Relaxations to graded low mechanical distension (< or =1 g) were insensitive to tetrodotoxin (TTX: 1 microM) and L-NOARG (300 microM), while those to higher distensions (2 g) were slightly inhibited by both agents to the same extent ( approximately 25%). In the human gastric fundus, NOS- and VIP immunoreactivities are colocalized in the majority of myenteric neurones. NO and VIP mediate electrically evoked relaxations: low frequency stimulation, irrespective of the duration, caused NO release only, whereas shortlasting stimulation at high frequencies induced NO and VIP release. Relaxations to graded mechanical distension were mostly due to passive viscoelastic properties, with a slight NO-mediated neurogenic component at 2 g distension. The difference between NO and VIP release suggests that in human fundus accommodation is initiated by NO. British Journal of Pharmacology (2000) 129, 12 - 20

Figure 1

Representative examples of the VIP innervation in the human gastric fundus. (a) VIP-IR varicose processes and thin bundles of fibres (arrows) and (b) thick bundles of processes throughout the muscle layer. (c) VIP-IR in a ganglion cell body (arrow) and fibres in the myenteric plexus. Calibration bar=25 μm.

Figure 2

NOS-IR in the human gastric fundus. Similarly to the VIP innervation, NOS labelled varicose nerve fibres (arrows) were densely distributed to the muscle layer of the gastric fundus (a), as well as in the myenteric plexus where they surrounded immunostained perikarya (arrows) (b). Calibration bar=25 μm.

Figure 3

Representative photomicrographs showing colocalization of VIP-IR (a) and NADPH-diaphorase histochemistry (b) in neurons (arrows) of the myenteric plexus of the human gastric fundus. Calibration bar=25 μm.

Figure 4

A representative tracing illustrating the motor response of a human isolated gastric fundus strip to electrical stimulation under control conditions or in the presence of 300 μM L-NOARG. Repetitive trains of electrical pulses at 1–50 Hz and 5 s in duration were delivered at 5 min intervals at 0.5 ms pulse width and 60 V. W indicates washing. Note the increase in tone, the reduction of NANC relaxations and the appearance of contractions induced by L-NOARG.

Figure 5

Frequency-dependent relaxant and contractile responses of gastric fundus strips to electrical stimulation, under control conditions or in the presence of L-NOARG. Values are expressed as per cent of the absolute value of the control inhibitory response obtained at 50 Hz and represent the means±s.e.mean of ten preparations. ^*P<0.05 versus control responses.

Figure 6

Tracing illustrating the effect of L-NOARG on electrically-evoked relaxations induced by stimulation at 1 Hz for 5 min. Note that L-NOARG (300 μM) increased the basal tone and turned the relaxant response into a contractile one.

Figure 7

Frequency-dependent contractile responses of gastric fundus strips to electrical stimulation in the presence of L-NOARG alone, in combination with hyoscine, or in combination with hyoscine plus TTX. Values are expressed as per cent of the maximal contraction obtained at 50 Hz in the presence of L-NOARG and represent the means of six preparations. For clarity, error bars (±s.e.mean) are included in the L-NOARG curve only. ^*P<0.05 versus L-NOARG alone.

Figure 8

Tracing illustrating the effect of VIP desensitization on L-NOARG (300 μM)-resistant electrically-induced NANC relaxations. Note the relaxation induced by the priming VIP (100 nM) administration, the lack of response following the second VIP administration, and the disappearance of NANC relaxations.

Figure 9

Frequency-dependent relaxations of gastric fundus strips to electrical stimulation under control conditions or after pharmacological treatment. Relaxations in control and following L-NOARG alone are data from Figure 5 (refer to this figure for statistical comparisons). The data with L-NOARG plus suramin or L-NOARG plus VIP desensitization are derived from five preparations each. Values are expressed as per cent of the relaxation obtained at 50 Hz. ^*P<0.05 versus L-NOARG alone.

Figure 10

Relaxations of gastric fundus strips to graded distensions under control conditions or in the presence of L-NOARG or TTX. Values are expressed as per cent of the maximal relaxation induced by 2 g load and represent the means±s.e.mean of 4–9 preparations. ^*P<0.05 versus control.